Serum albumins provide valuable scaffolds to which bioactive molecules may be fused, either through genetic fusions or chemical fusions to improve the properties of the fused molecule(s) (Leger, R. et al. (2004), Bioorg Med Chem Lett 14(17): 4395-8; Thibaudeau, K., et al. (2005). Bioconjug Chem 16(4): 1000-8; Balan, V. et al. (2006), Antivir Ther 11(1): 35-45; EP 0413622; WO 90/13653; EP 1681304; WO 1997/024445). Albumin has a long plasma half-life of about 19 days and because of this property it has been suggested for use in drug delivery.
The human serum albumin (HSA) polypeptide chain has 35 cysteine residues, which form 17 disulphide bonds and one unpaired (free) cysteine at position 34 of the mature protein (SEQ ID NO. 2). Cysteine-34 has been used for conjugation of molecules to albumin (Leger et al. (2004) Bioorg Med Chem Lett 14(17): 4395-8; Thibaudeau et al. (2005), Bioconjug Chem 16(4): 1000-8), and provides a precise, well defined site for conjugation. However, conjugation at cysteine-34 provides only one site for attachment of a single moiety and thus there is no choice of conjugation site. Also, the provision of a single conjugation site means that only one moiety can be conjugated to each albumin molecule. WO 2009/126920 and WO 2010/059315 propose the substitution for cysteine of one or more (e.g. several) selected surface-exposed threonine or serine residues in albumin. However, the actual production of such variants is not disclosed. WO 2010/092135 discloses albumin variants comprising three or more (several) conjugation-competent cysteine residues: cysteine-34 and at least two further cysteine residues; or variants in which another amino acid is substituted for the cysteine-34, and there are at least three further free cysteines.
Pharmaceutical agents, or their precursors, are generally prepared as homogeneous species, to allow for quality control. In HSA, the free cysteine at position 34 is located in a hydrophobic crevice with a depth of 9.5 Å (Cornell C N, Chang R, Kaplan L J. 1981. Arch. Biochem. Biophys. 209(1):1-6), and is not thought to be involved in homodimerization of HSA. However, surface-exposed cysteine residues in polypeptides may form stable inter-molecular disulphide bridges, as occur naturally for example between the heavy and light chains of immunoglobulin. It is desirable to provide albumin variants having introduced cysteine residues which have a low propensity to form dimers or oligomers.
WO 2000/69902 discloses conjugation of pharmaceutically beneficial compounds to HSA at cysteine-34, and it was found that the conjugates maintained the long plasma half-life of albumin. The resulting plasma half-life of the conjugate was generally considerably longer than the plasma half-life of the beneficial therapeutic compound alone. Further, albumin has been genetically fused to therapeutically beneficial peptides (WO 2001/79271A and WO 2003/59934) with the typical result that the fusion has the activity of the therapeutically beneficial peptide and a considerably longer plasma half-life than the plasma half-life of the therapeutically beneficial peptide alone.
Albumin binds in vivo to its receptor, the neonatal Fc receptor (FcRn) “Brambell” and this interaction is known to be important for the plasma half-life of albumin. FcRn is a membrane bound protein, expressed in many cell and tissue types. FcRn has been found to salvage albumin from intracellular degradation (Roopenian D. C. and Akilesh, S. (2007), Nat. Rev. Immunol 7, 715-725). FcRn is a bifunctional molecule that contributes to maintaining a high level of IgGs and albumin in plasma in mammals such as humans. Data indicate that IgG and albumin bind non-cooperatively to distinct sites on FcRn (Andersen et al. (2006), Eur. J. Immunol 36, 3044-3051; Chaudhury et al. (2006), Biochemistry 45, 4983-4990). Andersen et al. (2010), Journal of Biological Chemistry 285(7): 4826-36, describes the affinity of human and mouse FcRn for each of mouse and human albumin (all possible combinations). No binding of albumin from either species was observed at physiological pH to either receptor. At acidic pH, a 100-fold difference in binding affinity was observed.
The major FcRn receptor binding site in albumin is localized within Domain III (DIII, 381-585), (Andersen et al. (2010), Clinical Biochemistry 43, 367-372). A number of key amino acid residues have been shown to be important in binding, notably histidines H464, H510 and H536 and lysine K500 of human albumin (Andersen et al. (2010), Nat. Commun. 3:610. DOI:10.1038/ncomms1607). Generally, the higher the affinity of an albumin for FcRn, the longer is its plasma half-life. WO 2011/124718 discloses a class of variant albumins having modulated binding affinity to FcRn; the variants comprise domain III of an albumin with one or more (e.g. several) other domains of albumin and optionally include one or more (e.g. several) point mutations. WO 2012/059486 discloses variants of albumin in which a C-terminal portion of Domain III is swapped with a corresponding portion of an albumin of a different animal species. WO 2013/075066, WO2011/103076, WO 2012/112188, WO2011/051489 and WO 2014/072481 disclose point mutations within Domain III, or combinations of such point mutations, which alter the binding affinity of albumin to FcRn.
Various amino acid residues of albumin located in Domain I or Domain II have also recently been found to affect its interaction with FcRn. WO 2013/135896 discloses albumin variants having one or more (e.g. several) alterations in Domain I and one or more (e.g. several) alterations in Domain III. WO 2015/036579 discloses albumin variants having one or more (e.g. several) alterations in Domain II.
The listing or discussion of an apparently prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.
It is desirable to provide albumin variants having one or more (e.g. several) introduced cysteine residues in which an introduced free cysteine residue does not itself have a major impact on FcRn binding of albumin, or be positioned such that conjugation of a partner molecule to the free cysteine will sterically hinder FcRn binding. Such considerations could reduce the risk of unpredictable effects when introducing combinations of more than one free cysteine in a single albumin variant. Such variant polypeptides may be further modified to include alterations known to affect the binding affinity of albumin for FcRn, so as to allow the plasma half-life of the polypeptide, or conjugates thereof, to be tailored for specific applications.